Lactic Acid Bacteria Strains Used as Starters for Kimchi Fermentation Protect the Disruption of Tight Junctions in the Caco-2 Cell Monolayer Model

In this study, we investigated the effect of lactic acid bacteria (LAB) strains used as starters for kimchi fermentation, namely Lactococcus lactis WiKim0124, Companilactobacillus allii WiKim39, Leuconostoc mesenteroides WiKim0121 Leuconostoc mesenteroides WiKim33, and Leuconostoc mesenteroides WiKim32, on the intestinal epithelial tight junctions (TJs). These LAB strains were not cytotoxic to Caco-2 cells at 500 μg/ml concentration. In addition, hydrogen peroxide (H2O2) decreased Caco-2 viability, but the LAB strains protected the cells against H2O2-induced cytotoxicity. We also found that lipopolysaccharide (LPS) promoted Caco-2 proliferation; however, no specific changes were observed upon treatment with LAB strains and LPS. Our evaluation of the permeability in the Caco-2 monolayer model confirmed its increase by both LPS and H2O2. The LAB strains inhibited the increase in permeability by protecting TJs, which we evaluated by measuring TJ proteins such as zonula occludens-1 and occludin, and analyzing them by western blotting and immunofluorescence staining. Our findings show that LAB strains used for kimchi fermentation can suppress the increase in intestinal permeability due to LPS and H2O2 by protecting TJs. Therefore, these results suggest the possibility of enhancing the functionality of kimchi through its fermentation using functional LAB strains.


Immunofluorescence Staining
The LAB strains were incubated with either LPS (100 μg/ml for 24 h) or H 2 O 2 (500 μM for 5 h) on the monolayer. The cells were fixed with 4% paraformaldehyde for 15 min and blocked with 3% bovine serum albumin for 1 h. After the cells were rinsed with DPBS, primary antibodies, such as ZO-1 (1:100, 61-7300, Thermo Fisher Scientific, USA) and occludin (1:100, 42-2400, Thermo Fisher Scientific), were added and incubated at 4°C overnight. After rinsing with phosphate-buffered saline with Tween 20 (PBST) three times, the samples were incubated with Alexa Fluor 488-labeled secondary antibodies (1:1000) for 1 h at room temperature. Cell nuclei were stained by DAPI. Finally, the cells were observed under a fluorescence microscope after dropping the FluoromountTM Aqueous Mounting Medium (Sigma-Aldrich).

Western Blot Assay
The cells were harvested using RIPA buffer containing a 1% protease inhibitor. Then, they were homogenized using Bandelin ultrasonic homogenizer, and the homogenates were centrifuged at 13,000 ×g for 10 min at 4°C. The supernatants were used in the experiments. Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred to a polyvinylidene difluoride membrane. The membranes were incubated with primary antibodies (ZO-1 and occludin) overnight at 4°C. After rinsing with PBST three times, the membrane was incubated with a secondary antibody (1:1000) solution for 1 h at room temperature. The membrane was then treated with a chemiluminescence reagent and rinsed with PBST three times. The bands were observed using Gel Doc XR+ equipment (Bio-Rad Laboratories, Inc., USA).

Statistical Analysis
Data are represented as mean ± SD. Tukey's honest significant difference (HSD) test was carried out using the "Agricolae" package of the R software (v3.3.2; https://www.r-project.org/) for group comparisons.

Effect of LAB Strains on Caco-2 Cells
The LAB strains, except for WiKim0124, posed cytotoxicity to Caco-2 cells at a concentration of 1 mg/ml, and no other LAB strain showed cytotoxicity at 500 μg/ml concentration (Fig. 1A). Treatment with LPS and H 2 O 2 induced Caco-2 cell proliferation and cytotoxicity, respectively (Figs. 1B and 1C). According to Lin et al. (2015), LPS promotes cellular growth via c-Src upregulation in Caco-2 cells [16]. Accordingly, the tendency to increase cell viability was confirmed in the LPS-treated group, and there was no notable change in the treatment of LAB strains to Caco-2 cells treated with LPS (Fig. 1C). However, treatment with H 2 O 2 reduced cell viability by approximately 29% (Fig. 1B). H 2 O 2 rapidly kills any cell type through the oxidation of proteins, membrane lipids, and DNA by producing highly reactive hydroxyl radicals [17]. However, this oxidative damage can be inhibited by antioxidant activity.
Increased intake of exogenous antioxidants would alleviate the damage caused by oxidative stress by inhibiting the initiation or propagation of oxidative chain reaction, acting as free radical scavengers, quenchers of singlet oxygen, and reducing agents [18]. Natural antioxidants are widely distributed in food and medicinal plants [19]. The biological effects of probiotics are strain-specific, and although the success of one strain cannot be extrapolated to another strain, several studies have confirmed the antioxidant properties of various LAB [20][21][22][23]. In addition, WiKim0124 and WiKim39 strains were reported in a previous study to have antioxidant activity [24].

Effect of LAB Strains on Caco-2 Monolayer Permeability
Altered intestinal permeability influences many pathological conditions. The critical roles of intestinal permeability and barrier in health have been consistently confirmed [25]. In our experiments, both LPS and H 2 O 2 increased permeability (Fig. 2). Although the mechanisms may differ, LPS and H 2 O 2 were previously reported to increase permeability [26,27]. WiKim0124, at a treatment concentration of 500 μg/ml, was most effective in suppressing the increased permeability due to H 2 O 2 . These results are presumed to be related to the relatively higher protective effect of WiKim0124 against H 2 O 2 -induced cytotoxicity (Fig. 1B). H 2 O 2 induces oxidative stress, resulting in the loss of membrane integrity, increased permeability, and defective membrane transport mechanisms through oxidative degradation of membrane lipids [28]. Our findings indicate the antioxidant activity of WiKim0124 in mitigating H 2 O 2 toxicity.
WiKim33 and WiKim32 were relatively effective in inhibiting the LPS-induced increase in permeability (Fig. 2B). Initially, LPS was not toxic to cells but promoted cell differentiation (Fig. 1C); however, LPS significantly increased permeability (Fig. 2B). Permeability is controlled by endothelial cell substructural components reacting with a multitude of vasoactive cytokines, signal modulators, and growth factors. LPS induces the loss of the junction barrier through the activation of protein tyrosine kinases (PTKs) and caspase cleavage reactions [29]. In addition, LPS causes an increase in intestinal permeability via an intracellular mechanism involving TLR-4- dependent upregulation of CD14 membrane expression [30]. Here, the LAB strains effectively suppressed the increased LPS-induced permeability, and although the mechanism underlying the efficacy of LAB strains is yet to be elucidated, various LAB strains are regularly being reported to improve intestinal permeability effectively in in vivo and in vitro models [31][32][33]. In addition, probiotics reduce intestinal permeability in human patients [34]. More detailed research is needed, but nevertheless, our study demonstrates that WiKim0124 is effective in inhibiting the disruption of TJs due to oxidative stress, and further, that WiKim32 and WiKim33 are effective in inhibiting the disruption of TJs due to LPS.

Effect of LAB Strains on TJ Proteins
The human intestinal epithelium is formed by a single layer of epithelial cells. The space between these cells is sealed by TJs, which regulate the permeability of the intestinal barrier and act as a barrier between epithelial and endothelial cells [8,35]. A decrease in TJ proteins, such as ZO-1 and occludin, was confirmed in the LPS-and H 2 O 2 -treated groups with increased permeability (Figs. 3A and 4A). As a result of observing ZO-1 and occludin proteins through immunofluorescence staining, the destruction of TJ proteins was also observed in the LPS-and H 2 O 2 -treated groups (Figs. 3B and 4B). H 2 O 2 causes a loss of occludin and ZO-1 junctional localization, with subsequent punctuated staining at TJs, and causes stress fiber formation [36]. H 2 O 2 -induced decrease in electrical resistance and increase in inulin permeability are associated with the dephosphorylation of occludin on threonine residues [37]. In addition, claudins and occludins are often targeted and misplaced by viruses [38,39], bacteria [40,41], and inflammatory cytokines [41,42]. Furthermore, LPS reduces the expression of TJ proteins such as  occludin and ZO-1 [43].
LAB strains inhibited the reduction in TJ proteins caused by LPS or H 2 O 2 treatment (Figs. 3A and 4A), supporting the hypothesis that intestinal permeability was improved in the group of LAB strains treated with LPS or H 2 O 2 . However, studies on bioactive substances or related mechanisms of these effects of LAB strains remain lacking. Studies have only reported that some LAB strains increase the expression of TJ proteins. Specific Lactobacillus species abate barrier disruption by upregulating TJ protein expression, while L. acidophilus and L. plantarum have increased occludin protein expression in in vivo and in vitro models, respectively [31,32]. The beneficial effects of probiotics on intestinal barrier function are at least partially mediated through organismal stimulation of toll-like receptors (TLRs), particularly TLR2, and sequential changes in TJ protein expression and localization [12]. Although it is necessary to confirm the exact mechanism of improvement of each strain for these TJ changes, this study is meaningful in confirming that strains used for kimchi fermentation positively affect intestinal permeability.
In conclusion, LAB strains used for kimchi fermentation effectively alleviate the disruption of intestinal permeability in the Caco-2 cell monolayer model. These LAB strains had a protective effect against Caco-2 cytotoxicity and increased permeability caused by H 2 O 2 . In addition, although LPS was not toxic to cells, it increased permeability. LAB strains inhibited the LPS-induced permeability by inhibiting the reduction of TJ proteins such as ZO-1 and occludin caused by LPS and H 2 O 2 . Further and more diverse studies are necessary to investigate not only the mechanisms behind the functionality of LAB strains, but also to identify other strains with highly beneficial activities. Although our study did not identify such a strain, our findings should stimulate the future development of the kimchi starter culture industry.